Certain nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection. However, HBV often acquires NRTI resistance, making it crucial to develop novel agents that offer more profound suppression of wild-type HBV and drug-resistant variants. Here we report a newly designed and synthesized fluorine-containing NRTI, CFCP. CFCP potently blocked the production of HBV (EC50 3 nM) in HepG2.5.5.15.7 cells without significant cytotoxicity (CC50 100 microM in HepG2 cells). CFCP also blocked the infectivity and replication of wild-type HBV (HBVWTCe) more potently (EC50 value being 0.39 nM) than entecavir (ETV; EC50=3.4 nM) as assessed with Southern blot assay using HBVWTCe-exposed Huh7 cells. CFCP also exerted potent activity against ETV-resistant HBVETV-RL180M/S202G/M204V with an EC50 value being 65.3 nM, while EC50 values of ETV, adefovir (ADV), and tenofovir (TDF) were 1,000, 30,775, and 406.6 nM, respectively. Moreover, CFCP was potent against ADV-resistant HBVADV-RA181T/N236T with an EC50 value being 7.0 nM, while EC50 values of ETV, ADV, and TDF against HBVADV-RA181T/N236T were 184.7, 120,870, and 218.5 nM, respectively. CC50 values of CFCP to MT2 cells, HepG2 cells, and human hepatocytes (PXB cells) were 400, 50, and 100 microM, while CC50 values of ETV were 126, 53, and 100 microM, respectively. Once daily peroral administration of CFCP over 2 weeks (0.2 mg/kg/day) reduced HBVWTCe viremia by 2-3 logs in human-liver-chimeric/HBVWTCe-infected mice and the reduction persisted over 2-3 weeks following treatment cessation. CFCP also reduced HBVETV-RL180M/S202G/M204V viremia by 2 logs in human-liver-chimeric/HBVETV-RL180M/S202G/M204V-infected mice, while ETV (0.2 mg/kg) completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. Structural analysis revealed that although ETV-TP forms good van der Waals contacts with L180 and M204 of HBVWTCe's reverse transcriptase (RT), those contacts are lost in HBVETV-RL180M/S202G/M204V's RT. However, CFCP-TP has good contacts with RT of both HBVWTCe and HBVETV-RL180M/S202G/M204V, to which CFCP-TP's fluorine atom and cyano moiety greatly contributes. None of the CFCP (up to 5 mg/kg/day)-administered mice had significant drug-related body-weight or serum human-albumin concentration changes and no histological abnormalities were seen in liver, heart, lung, or kidney. The present data warrant that further study be conducted toward potential clinical development of CFCP as a therapeutic for infection with drug-resistant HBV variants. Moreover, we are currently designing and newly synthesizing analogs of CFCP in hope to obtain back-up compounds and furthermore potent and HBV's resistance-repellant compounds.